Pyrazino 1&#39;,2&#39;:1,6 pyrido 3,4-b indole1,4-dione derivatives

ABSTRACT

Compounds of the general structural formula (I), and use of the compounds and salts and solvates thereof, as thereapeutic agents. In particular, the invention relates to compounds that are potent and selective inhibitors of cyclic guanosine 3′, 5′-monophosphate specific phosphodiesterase (cGMP-specific PDE), in particular PDE5, and have utility in a variety of therapeutic areas wherein such inhibition is considered beneficial, including the treatment of cardiovascular disorders and erectile dysfunction.

FIELD AND BACKGROUND OF THE INVENTION

[0001] This invention relates to a series of compounds, to methods ofpreparing the compounds, to pharmaceutical compositions containing thecompounds, and to their use as therapeutic agents. In particular, theinvention relates to compounds that are potent and selective inhibitorsof cyclic guanosine 3′,5′-monophosphate specific phosphodiesterase(cGMP-specific PDE), in particular PDE5, and have utility in a varietyof therapeutic areas wherein such inhibition is considered beneficial,including the treatment of cardiovascular disorders and erectiledysfunction.

SUMMARY OF THE INVENTION

[0002] The present invention provides compounds of formula (I)

[0003] wherein R⁰, independently, is selected from the group consistingof halo and C₁₋₆alkyl;

[0004] R¹ is selected from the group consisting of hydro, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, haloC₁₋₆-alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, aryl-C₁₋₃alkyl, C₁₋₃alkylenearyl, andheteroarylC₁₋₃alkyl;

[0005] R² is selected from the group consisting of an optionallysubstituted monocyclic aromatic ring selected from the group consistingof benzene, thiophene, furan, and pyridine, and an optionallysubstituted bicyclic ring

[0006] wherein the fused ring A is a 5- or 6-membered ring, saturated orpartially or fully unsaturated, and comprises carbon atoms andoptionally one or two heteroatoms selected from oxygen, sulfur, andnitrogen;

[0007] R³ is selected from the group consisting of hydro and C₁₋₆alkyl,

[0008] or R¹ and R³ together form a 3- or 4-membered alkyl or alkenylchain component of a 5- or 6-membered ring;

[0009] R⁴ is selected from the group consisting of C₁₋₆alkyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, C₂₋₆-alkenyl, C₁₋₃alkylenearyl,arylC₁₋₃alkyl, heteroaryl-C₁₋₃alkyl, C(═O)R^(a), aryl, heteroaryl,C(═O)OR^(a), C(═O)—NR^(a)R^(b), C(═S)NR^(a)R^(b), SO₂R^(a), SO₂OR^(a),SO₂NR^(a)R^(b), S(═O)R^(a), S(═O)NR^(a)R^(b),C(═O)NR^(a)C₁₋₄alkyleneOR^(a), C(═O)NR^(a)C₁₋₄alkyleneHet,C(═O)C₁₋₄alkylenearyl, C(═O)C₁₋₄alkylenehetero-aryl, C₁₋₄alkylenearylsubstituted with one or more of SO₂NR^(a)R^(b), NR^(a)R^(b),C(═O)OR^(a), NR^(a)SO₂CF₃, CN, NO₂, C(═O)R^(a), OR^(a),C₁₋₄alkyleneNR^(a)R^(b), and OC₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)C₁₋₄alkylenearyl,C₁₋₄alkyleneC(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkylene-C(═O)Het,C₁₋₄alkyleneC(═O)NR^(a)R^(b), C₁₋₄alkyleneOR^(a),C₁₋₄alkyleneNR^(a)C(═O)R^(a), C₁₋₄alkyleneOC₁₋₄alkyleneOR^(a),C₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneC(═O)OR^(a), andC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a);

[0010] Het represents a 5- or 6-membered heterocyclic ring, saturated orpartially or fully unsaturated, containing at least one heteroatomselected from the group consisting of oxygen, nitrogen, and sulfur, andoptionally substituted with C₁₋₄alkyl or C(═O)OR^(a);

[0011] R^(a) is selected from the group consisting of hydro, C₁₋₆alkyl,aryl, arylC₁₋₃alkyl, C₁₋₃alkylenearyl, heteroaryl, heteroarylC₁₋₃alkyl,and C₁₋₃alkyleneheteroaryl;

[0012] R^(b) is selected from the group consisting of hydro, C₁₋₆alkyl,aryl, heteroaryl, arylC₁₋₃alkyl, heteroarylC₁₋₃alkyl,C₁₋₃alkyleneN(R^(a))₂, C₁₋₃alkylenearyl, C₁₋₃alkyleneHet, haloC₁₋₃alkyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, C₁₋₃alkyleneheteroaryl,C₁₋₃-alkyleneC(═O)OR^(a), and C₁₋₃alkyleneC₃₋₈heterocycloalkyl;

[0013] or R^(a) and R^(b) are taken together to form a 5- or 6-memberedring, optionally containing at least one heteroatom;

[0014] q is 0, 1, 2, 3, or 4; and

[0015] pharmaceutically acceptable salts and hydrates thereof.

[0016] As used herein, the term “alkyl” includes straight chained andbranched hydrocarbon groups containing the indicated number of carbonatoms, typically methyl, ethyl, and straight chain and branched propyland butyl groups. The hydrocarbon group can contain up to 16 carbonatoms. The term “alkyl” includes “bridged alkyl,” e.g., a C₆-C₁₆bicyclic or polycyclic hydrocarbon group, for example, norbornyl,adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl,bicyclo[3.2.1]octyl, and decahydronaphthyl.” The term “cycloalkyl” isdefined as a cyclic C₃-C₈ hydrocarbon group, e.g., cyclopropyl,cyclobutyl, cyclohexyl, and cyclopentyl.

[0017] The terms “alkenyl” and “alkynyl” are defined identically as“alkyl,” except for containing a carbon-carbon double bond orcarbon-carbon triple bond, respectively. “Cycloalkenyl” is definedsimilarly to cycloalkyl, except a carbon-carbon double bond is presentin the ring.

[0018] The term “alkylene” refers to an alkyl group having asubstituent. For example, the term “C₁₋₃alkylenearyl” refers to an alkylgroup containing one to three carbon atoms, and substituted with an arylgroup.

[0019] The term “halo” or “halogen” is defined herein to includefluorine, bromine, chlorine, and iodine.

[0020] The term “haloalkyl” is defined herein as an alkyl groupsubstituted with one or more halo substituents, either fluoro, chloro,bromo, or iodo. Similarly, “halocycloalkyl” is defined as a cycloalkylgroup having one or more halo substituents.

[0021] The term “aryl,” alone or in combination, is defined herein as amonocyclic or polycyclic aromatic group, preferably a monocyclic orbicyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwiseindicated, an “aryl” group can be unsubstituted or substituted, forexample, with one or more, and in particular one to three, halo, alkyl,hydroxy, C(═O)OR^(a), hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl,haloalkoxy, cyano, nitro, amino, alkylamino, acylamino, alkylthio,alkylsulfinyl, and alkylsulfonyl. Exemplary aryl groups include phenyl,naphthyl, tetrahydronaphthyl, 2-chlorophenyl, 3-chlorophenyl,4-chlorophenyl, 2-methylphenyl, 4-methoxyphenyl,3-trifluoromethylphenyl, 4-nitrophenyl, and the like. The terms“arylC₁₋₃alkyl” and “heteroarylC₁₋₃alkyl” are defined as an aryl orheteroaryl group having a C₁₋₃alkyl substituent.

[0022] The term “heteroaryl” is defined herein as a monocyclic orbicyclic ring system containing one or two aromatic rings and containingat least one nitrogen, oxygen, or sulfur atom in an aromatic ring, andwhich can be unsubstituted or substituted, for example, with one ormore, and in particular one to three, substituents, like halo, alkyl,hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino,alkylamino, acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl.Examples of heteroaryl groups include thienyl, furyl, pyridyl, oxazolyl,quinolyl, isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl,imidizolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, andthiadiazolyl.

[0023] The term “Het” is defined as monocyclic, bicyclic, and tricyclicgroups containing one or more heteroatoms selected from the groupconsisting of oxygen, nitrogen, and sulfur. A “Het” group also cancontain an oxo group (═O) attached to the ring. Nonlimiting examples ofHet groupsinclude 1,3-dioxolanyl dioxolanyl, 2-pyrazolinyl,pyrazolidinyl, pyrrolidinyl, piperazinyl, a pyrrolinyl, 2H-pyranyl,4H-pyranyl, morpholinyl, thiopholinyl, piperidinyl, 1,4-dithianyl, and1,4-dioxane.

[0024] The term “hydroxy” is defined as —OH.

[0025] The term “alkoxy” is defined as —OR, wherein R is alkyl.

[0026] The term “alkoxyalkyl” is defined as an alkyl group wherein ahydrogen has been replaced by an alkoxy group. The term“(alkylthio)alkyl” is defined similarly as alkoxyalkyl, except a sulfuratom, rather than an oxygen atom, is present.

[0027] The term “hydroxyalkyl” is defined as a hydroxy group appended toan alkyl group.

[0028] The term “amino” is defined as —NH₂, and the term “alkylamino” isdefined as —NR₂, wherein at least one R is alkyl and the second R isalkyl or hydrogen.

[0029] The term “acylamino” is defined as RC(═O)N, wherein R is alkyl oraryl.

[0030] The term “alkylthio” is defined as —SR, wherein R is alkyl.

[0031] The term “alkylsulfinyl” is defined as R—SO₂, wherein R is alkyl.

[0032] The term “alkylsulfonyl” is defined as R—SO₃, wherein R is alkyl.

[0033] The term “nitro” is defined as —NO₂.

[0034] The term “trifluoromethyl” is defined as —CF₃.

[0035] The term “trifluoromethoxy” is defined as —OCF₃.

[0036] The term “cyano” is defined as —CN.

[0037] In a preferred embodiment, q is 0, or R⁰ is selected from thegroup consisting of halo and C₁₋₃alkyl.

[0038] In a preferred group of compounds of formula (I), R² isrepresented by

[0039] wherein the bicyclic ring can represent, for example, naphthaleneor indene, or a heterocycle, such as benzoxazole, benzothiazole,benzisoxazole, benzimidazole, quinoline, indole, benzothiophene, orbenzofuran, or

[0040] wherein n is an integer 1 or 2, and G, independently, isC(R^(a))₂, O, S, or NR^(a). The bicyclic ring comprising the R²substituent typically is attached to the rest of the molecule by aphenyl ring carbon atom.

[0041] In another preferred group of compounds of formula (I), R² isrepresented by an optionally substituted bicyclic ring

[0042] wherein n is 1 or 2, and G, independently, are C(R^(a))₂ or O.Especially preferred R² substituents include

[0043] Within this particular group of compounds, nonlimiting examplesof substituents for the bicyclic ring include halo (e.g., chloro),C₁₋₃alkyl (e.g., methyl, ethyl, or i-propyl), OR^(a) (e.g., methoxy,ethoxy, or hydroxy), CO₂R^(a), halomethyl or halomethoxy (e.g.,trifluoromethyl or trifluoromethoxy), cyano, nitro, and NR^(a)R^(b).

[0044] In a preferred embodiment, R⁴ is selected from the groupconsisting of C₁₋₆alkyl, aryl, heteroaryl, C(═O)R^(a), SO₂NR^(a)R^(b),C(═O)OR^(a), C₁₋₄alkyleneHet, C₁₋₄alkyleneheteroalyl, C₁₋₄alkylenearyl,C₁₋₄alkyleneC(═O)C₁₋₄alkylenearyl, C₁₋₄alkyleneC(═O)OR^(a),C₁₋₄alkyleneC(═O)NR^(a)R^(b), C₁₋₄alkyleneC(═O)Het,C₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneOR^(a) andC₁₋₄alkyleneNR^(a)C(═O)R^(a).

[0045] In more preferred embodiments, R⁴ is selected from the groupconsisting of C₁₋₆alkyl, C(═O)R^(a), SO₂NR^(a)R^(b), andC₁₋₄alkyleneHet, wherein Het is selected from the group consisting ofpiperazinyl, morpholinyl, pyrrolidinyl, pyrrolidonyl, tetrahydrofuran,piperidinyl,

[0046] C₁₋₄alkyleneC₆H₅, optionally substituted with one to three groupsselected from the group consisting of C(═O)OR^(a), NR^(a)R^(b),NR^(a)SO₂CF₃, SO₂NR^(a)R^(b), CN, OR^(a), C(═O)R^(a),C₁₋₄alkyleneNR^(a)R^(b), nitro, OC₁₋₄alkylenearyl, andOC₁₋₄alkyleneNR^(a)R^(b); C₁₋₄alkyleneOR^(a);C₁₋₄alkyleneC(═O)NR^(a)R^(b); C₁₋₄alkyleneC(═O)NR^(a)R^(c); C₆H₅;C₁₋₄alkyleneNR^(a)R^(b); and C₁₋₄alkyleneNHC(═O)R^(a).

[0047] In especially preferred embodiments, q is 0 or R⁰ is selectedfrom the group consisting of halo and methyl; R¹ is selected from thegroup consisting of hydro, C₁₋₆alkyl, and haloC₁₋₆alkyl; R² is selectedfrom the group consisting of

[0048] R³ is C₁₋₆alkyl; and R⁴ is selected from the group consisting ofCH₃, (CH₂)₄C(═O)OH, C(═O)OCH₃, C(═O)CH₃, CH₂NHCH₂C₆H₅, CH₂NH₂, CHO,C₂H₅, CH(CH₃)₂, CH₂OH, SO₂N(CH₃)₂, and

[0049] An especially preferred subclass of compounds within the generalscope of formula (I) is represented by compounds of formula (II)

[0050] and pharmaceutically acceptable salts and solvates (e.g.,hydrates) thereof.

[0051] Compounds of formula (I) can contain one or more asymmetriccenter, and, therefore, can exist as stereoisomers. The presentinvention includes both mixtures and separate individual stereoisomersof the compounds of formula (I). Compounds of formula (I) also can existin tautomeric forms, and the invention includes both mixtures andseparate individual tautomers thereof.

[0052] Pharmaceutically acceptable salts of the compounds of formula (I)can be acid addition salts formed with pharmaceutically acceptableacids. Examples of suitable salts include, but are not limited to, thehydrochloride, hydrobromide, sulfate, bisulfate, phosphate, hydrogenphosphate, acetate, benzoate, succinate, fumarate, maleate, lactate,citrate, tartrate, gluconate, methanesulfonate, benzenesulfonate, andp-toluenesulfonate salts. The compounds of formula (I) also can providepharmaceutically acceptable metal salts, in particular alkali metalsalts and alkaline earth metal salts, with bases. Examples include thesodium, potassium, magnesium, and calcium salts.

[0053] Compounds of the present invention are potent and selectiveinhibitors of cGMP-specific PDES. Thus, compounds of formula (I) are ofinterest for use in therapy, specifically for the treatment of a varietyof conditions where selective inhibition of PDE5 is considered to bebeneficial.

[0054] Phosphodiesterases (PDEs) catalyze the hydrolysis of cyclicnucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclicguanosine monophosphate (cGMP). The PDEs have been classified into atleast seven isoenzyme families and are present in many tissues (J. A.Beavo, Physiol. Rev., 75, p. 725 (1995)).

[0055] PDE5 inhibition is a particularly attractive target. A potent andselective inhibitor of PDE5 provides vasodilating, relaxing, anddiuretic effects, all of which are beneficial in the treatment ofvarious disease states. Research in this area has led to several classesof inhibitors based on the cGMP basic structure (E. Sybertz et al.,Expert. Opin. Ther. Pat., 7, p. 631 (1997)).

[0056] The biochemical, physiological, and clinical effects of PDE5inhibitors therefore suggest their utility in a variety of diseasestates in which modulation of smooth muscle, renal, hemostatic,inflammatory, and/or endocrine function is desirable. The compounds offormula (I), therefore, have utility in the treatment of a number ofdisorders, including stable, unstable, and variant (Prinzmetal) angina,hypertension, pulmonary hypertension, congestive heart failure, chronicobstructive pulmonary disease, malignant hypertension, pheochromocytoma,acute respiratory distress syndrome, congestive heart failure, acute andchronic renal failure, atherosclerosis, conditions of reduced bloodvessel patency (e.g., postpercutaneous transluminal coronary or carotidangioplasty, or post-bypass surgery graft stenosis), peripheral vasculardisease, vascular disorders, such as Raynaud's disease, thrombocythemia,inflammatory diseases, myocardial infarction, stroke, bronchitis,chronic asthma, allergic asthma, allergic rhinitis, glaucoma,osteoporosis, preterm labor, benign prostatic hypertrophy, peptic ulcer,male erectile dysfunction, female sexual dysfunction, and diseasescharacterized by disorders of gut motility (e.g., irritable bowelsyndrome).

[0057] An especially important use is the treatment of male erectiledysfunction, which is one form of impotence and is a common medicalproblem. Impotence can be defined as a lack of power, in the male, tocopulate, and can involve an inability to achieve penile erection orejaculation, or both. The incidence of erectile dysfunction increaseswith age, with about 50% of men over the age of 40 suffering from somedegree of erectile dysfunction.

[0058] In addition, a further important use is the treatment of femalearousal disorder, also termed female sexual arousal disorder. Femalearousal disorders are defined as a recurrent inability to attain ormaintain an adequate lubrication/swelling response of sexual excitementuntil completion of sexual activity. The arousal response consists ofvasocongestion in the pelvis, vaginal lubrication, and expansion andswelling of external genitalia.

[0059] It is envisioned, therefore, that compounds of formula (I) areuseful in the treatment of male erectile dysfunction and female arousaldisorder. Thus, the present invention concerns the use of compounds offormula (I), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing either entity, for the manufactureof a medicament for the curative or prophylactic treatment of erectiledysfunction in a male animal and arousal disorder in a female animal,including humans.

[0060] The term “treatment” includes preventing, lowering, stopping, orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” includes both medical therapeuticand/or prophylactic administration, as appropriate.

[0061] It also is understood that “a compound of formula (I),” or aphysiologically acceptable salt or solvate thereof, can be administeredas the neat compound, or as a pharmaceutical composition containingeither entity.

[0062] Although the compounds of the invention are envisioned primarilyfor the treatment of sexual dysfunction in humans, such as male erectiledysfunction and female arousal disorder, they also can be used for thetreatment of other disease states.

[0063] A further aspect of the present invention, therefore, isproviding a compound of formula (I) for use in the treatment of stable,unstable, and variant (Prinzmetal) angina, hypertension, malignanthypertension, pheochromocytoma, pulmonary hypertension, chronicobstructive pulmonary disease, congestive heart failure, acuterespiratory distress syndrome, acute and chronic renal failure,atherosclerosis, conditions of reduced blood vessel patency (e.g.,post-PTCA or post-bypass graft stenosis), peripheral vascular disease,vascular disorders such as Raynaud's disease, thrombocythemia,inflammatory diseases, prophylaxis of myocardial infarction, prophylaxisof stroke, stroke, bronchitis, chronic asthma, allergic asthma, allergicrhinitis, glaucoma, osteoporosis, preterm labor, benign prostatichypertrophy, male and female erectile dysfunction, or diseasescharacterized by disorders of gut motility (e.g., IBS).

[0064] According to another aspect of the present invention, there isprovided the use of a compound of formula (I) for the manufacture of amedicament for the treatment of the above-noted conditions anddisorders.

[0065] In a further aspect, the present invention provides a method oftreating the above-noted conditions and disorders in a human or nonhumananimal body which comprises administering to said body a therapeuticallyeffective amount of a compound of formula (I).

[0066] Compounds of the invention can be administered by any suitableroute, for example by oral, buccal, inhalation, sublingual, rectal,vaginal, transurethral, nasal, topical, percutaneous, i.e., transdermal,or parenteral (including intravenous, intramuscular, subcutaneous, andintracoronary) administration. Parenteral administration can beaccomplished using a needle and syringe, or using a high pressuretechnique, like POWDERJEC™.

[0067] Oral administration of a compound of the invention is thepreferred route. Oral administration is the most convenient and avoidsthe disadvantages associated with other routes of administration. Forpatients suffering from a swallowing disorder or from impairment of drugabsorption after oral administration, the drug can be administeredparenterally, e.g., sublingually or buccally.

[0068] Compounds and pharmaceutical compositions suitable for use in thepresent invention include those wherein the active ingredient isadministered in an effective amount to achieve its intended purpose.More specifically, a “therapeutically effective amount” means an amounteffective to prevent development of, or to alleviate the existingsymptoms of, the subject being treated. Determination of the effectiveamounts is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

[0069] A “therapeutically effective dose” refers to that amount of thecompound that results in achieving the desired effect. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, which is expressed as the ratio between LD₅₀ andED₅₀. Compounds which exhibit high therapeutic indices are preferred.The data obtained from such data can be used in formulating a range ofdosage for use in humans. The dosage of such compounds preferably lieswithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed, and the route of administration utilized.

[0070] The exact formulation, route of administration, and dosage can bechosen by the individual physician in view of the patient's condition.Dosage amount and interval can be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thetherapeutic effects.

[0071] The amount of composition administered is dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration, and the judgment of theprescribing physician.

[0072] Specifically, for administration to a human in the curative orprophylactic treatment of the conditions and disorders identified above,oral dosages of a compound of formula (I) generally are about 0.5 toabout 1000 mg daily for an average adult patient (70 kg). Thus, for atypical adult patient, individual tablets or capsules contain 0.2 to 500mg of active compound, in a suitable pharmaceutically acceptable vehicleor carrier, for administration in single or multiple doses, once orseveral times per day. Dosages for intravenous, buccal, or sublingualadministration typically are 0.1 to 500 mg per single dose as required.In practice, the physician determines the actual dosing regimen which ismost suitable for an individual patient, and the dosage varies with theage, weight, and response of the particular patient. The above dosagesare exemplary of the average case, but there can be individual instancesin which higher or lower dosages are merited, and such are within thescope of this invention.

[0073] For human use, a compound of the formula (I) can be administeredalone, but generally is administered in admixture with a pharmaceuticalcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice. Pharmaceutical compositions for use inaccordance with the present invention thus can be formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries that facilitateprocessing of compounds of formula (I) into preparations which can beused pharmaceutically.

[0074] These pharmaceutical compositions can be manufactured in aconventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping, or lyophilizing processes. Proper formulation is dependentupon the route of administration chosen. When a therapeuticallyeffective amount of a compound of the present invention is administeredorally, the composition typically is in the form of a tablet, capsule,powder, solution, or elixir. When administered in tablet form, thecomposition can additionally contain a solid carrier, such as a gelatinor an adjuvant. The tablet, capsule, and powder contain about 5% toabout 95% compound of the present invention, and preferably from about25% to about 90% compound of the present invention. When administered inliquid form, a liquid carrier such as water, petroleum, or oils ofanimal or plant origin can be added. The liquid form of the compositioncan further contain physiological saline solution, dextrose or othersaccharide solutions, or glycols. When administered in liquid form, thecomposition contains about 0.5% to about 90% by weight of a compound ofthe present invention, and preferably about 1% to about 50% of acompound of the present invention.

[0075] When a therapeutically effective amount of a compound of thepresent invention is administered by intravenous, cutaneous, orsubcutaneous injection, the composition is in the form of a pyrogenfree,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable solutions, having due regard to pH, isotonicity,stability, and the like, is within the skill in the art. A preferredcomposition for intravenous, cutaneous, or subcutaneous injectiontypically contains, in addition to a compound of the present invention,an isotonic vehicle.

[0076] For oral administration, the compounds can be formulated readilyby combining a compound of formula (I) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the presentcompounds to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by adding a compound of formula (I) with asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients include, forexample, fillers and cellulose preparations. If desired, disintegratingagents can be added.

[0077] For administration by inhalation, compounds of the presentinvention are conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin, for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0078] The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing, and/or dispersing agents.

[0079] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils or synthetic fatty acid esters. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions. Alternatively, a present composition can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

[0080] Compounds of the present invention also can be formulated inrectal compositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases. In addition to theformulations described previously, the compounds also can be formulatedas a depot preparation. Such long-acting formulations can beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

[0081] Many of the compounds of the present invention can be provided assalts with pharmaceutically compatible counterions. Suchpharmaceutically acceptable base addition salts are those salts thatretain the biological effectiveness and properties of the free acids,and that are obtained by reaction with suitable inorganic or organicbases.

[0082] In particular, a compound of formula (I) can be administeredorally, buccally, or sublingually in the form of tablets containingexcipients, such as starch or lactose, or in capsules or ovules, eitheralone or in admixture with excipients, or in the form of elixirs orsuspensions containing flavoring or coloring agents. Such liquidpreparations can be prepared with pharmaceutically acceptable additives,such as suspending agents. A compound also can be injected parenterally,for example, intravenously, intramuscularly, subcutaneously, orintracoronarily. For parenteral administration, the compound is bestused in the form of a sterile aqueous solution which can contain othersubstances, for example, salts, or monosaccharides, such as mannitol orglucose, to make the solution isotonic with blood.

[0083] For veterinary use, a compound of formula (I) or a nontoxic saltthereof, is administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal.

[0084] Thus, the invention provides in a further aspect a pharmaceuticalcomposition comprising a compound of the formula (I), together with apharmaceutically acceptable diluent or carrier therefor. There isfurther provided by the present invention a process of preparing apharmaceutical composition comprising a compound of formula (I), whichprocess comprises mixing a compound of formula (I), together with apharmaceutically acceptable diluent or carrier therefor.

[0085] In a particular embodiment, the invention includes apharmaceutical composition for the curative or prophylactic treatment oferectile dysfunction in a male animal, or arousal disorder in a femaleanimal, including humans, comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable diluent or carrier.

[0086] Compounds of formula (I) can be prepared by any suitable methodknown in the art, or by the following processes which form part of thepresent invention. In the methods below, R⁰, R¹, R², R³, and R⁴, aredefined as in structural formula (I) above. In particular, compounds ofstructural formula (I) can be prepared according to the followingsynthetic schemes.

[0087] Several methods exist for synthesizing β-carbolines. For example,Daugan U.S. Pat. No. 5,859,006, incorporated herein by reference,discloses preparation of compounds of structural formulae (III) and (IV)

[0088] The compounds of structural formula (I) can be prepared in ananalogous manner as a compound of structural formula (IV) usingappropriately substituted starting materials.

[0089] Alternatively, a compound of structural formula (IV) can beprepared, then the indole nitrogen can be directly alkylated or acylatedto provide the desired R⁴ substituent. Preparation of N-substitutedindoles are well known to persons skilled in the art.

[0090] It should be understood that protecting groups can be utilized inaccordance with general principles of synthetic organic chemistry toprovide compounds of structural formula (I). Protecting group-formingreagents, like benzyl chloroformate and trichloroethyl chloroformate,are well known to persons skilled in the art, for example, see T. W.Greene et al., “Protective Groups in Organic Synthesis, Third Edition,”John Wiley and Sons, Inc., NY, N.Y. (1999). These protecting groups areremoved when necessary by appropriate basic, acidic, or hydrogenolyticconditions known to persons skilled in the art. Accordingly, compoundsof structural formula (I) not specifically exemplified herein can beprepared by persons skilled in the art.

[0091] In addition, compounds of formula (I) can be converted to othercompounds of formula (I), or a compound of structural formula (IV) canbe converted to a compound of structural formula (I). Thus, for example,a particular R substituent can be interconverted to prepare anothersuitably substituted compound of formula (I). Examples of appropriateinterconversions include, but are not limited to, OR^(a) to hydroxy bysuitable means (e.g., using an agent such as SnCl₂ or a palladiumcatalyst, like palladium-on-carbon), or amino to substituted amino, suchas acylamino or sulphonylamino, using standard acylating orsulfonylating conditions. Other interconversions include indole N—H toindole N—R⁴, nitro to amino, and cyano to C(═O)OR^(a) orC(═O)NR^(a)R^(b).

[0092] Compounds of formula (I) can be prepared by the method above asindividual stereoisomers or as a racemic mixture. Individualstereoisomers of the compounds of the invention can be prepared fromracemates by resolution using methods known in the art for theseparation of racemic mixtures into their constituent stereoisomers, forexample, using HPLC on a chiral column, such as Hypersil naphthyl urea,or using separation of salts of stereoisomers. Compounds of theinvention can be isolated in association with solvent molecules bycrystallization from, or evaporation of, an appropriate solvent.

[0093] The pharmaceutically acceptable acid addition salts of thecompounds of formula (I) that contain a basic center can be prepared ina conventional manner. For example, a solution of the free base can betreated with a suitable acid, either neat or in a suitable solution, andthe resulting salt isolated either by filtration or by evaporation undervacuum of the reaction solvent. Pharmaceutically acceptable baseaddition salts can be obtained in an analogous manner by treating asolution. of a compound of formula (I) with a suitable base. Both typesof salt can be formed or interconverted using ion-exchange resintechniques. Thus, according to a further aspect of the invention, amethod for preparing a compound of formula (I) or a salt or solvate(e.g., hydrate) is provided, followed by (i) salt formation, or (ii)solvate (e.g., hydrate) formation.

[0094] The following additional abbreviations are used hereafter in theaccompanying examples: rt (room temperature), min (minute), h (hour), g(gram), mmol (millimole), m.p. (melting point), eq (equivalents), L(liter), TL (milliliter), μL (microliter), DMSO (dimethyl sulfoxide),CH₂CL₂ (dichloromethane), IPA (isopropyl alcohol), MeOH (methanol), DMF(dimethylformamide), Ac₂O (acetic anhydride), Et₃N (triethylamine),MeNH₂ (methylamine), sat. (saturated), CH₃I (methyl iodide), NaH (sodiumhydride), NH₄Cl (ammonium chloride), Na₂SO₄ (sodium sulfate), EtOAc(ethyl acetate), SOCl₂ (thionyl chloride), Et₂O (diethyl ether), CHCl₃(chloroform), NaHSO₄ (sodium bisulfate), NaHCO₃ (sodium bicarbonate),HCl (hydrochloric acid), NaCl (sodium chloride), and THF(tetrahydrofuran).

EXAMPLE 1

[0095](6R,12aS)-6-Benzo[1,3]dioxol-5-yl-2,7-dimethyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indole-1,4-dione

[0096] Example 1 was prepared in one step from compound (IV) byalkylation with methyl iodide. Under basic reaction conditions, compound(IV) was completely epimerized at the C12a position.

[0097] A solution of compound (IV) (1.95 g, 5.0 mmol) in THF (60 mL)(prepared by dissolving compound (IV) in hot THF and cooled to roomtemperature) was added to a slurry of NaH (80% in mineral oil, 260 mg,9.1 mmol) in THF (10 mL) at 0° C. under a nitrogen blanket over a periodof 3 minutes. The mixture was stirred at 0° C. for 30 minutes afterwhich CH₃I (0.44 mL, 7.0 mmol) was added dropwise. The resulting mixturewas stirred at 5° C. for an additional 30 minutes after which themixture was diluted with CH₂Cl₂ (200 mL). The organic layer was washedsuccessively with sat. NH₄Cl (50 mL) and brine (20 mL), dried overNa₂SO₄, filtered, and the solvent was removed under reduced pressure toprovide a yellow solid. The residue was dissolved in a boiling mixtureof CH₂Cl₂ (15 mL), THF (30 mL), and methyl tert-butyl ether (20 mL),then the solution was filtered under vacuum while hot. The solidprecipitate from the filtrate was collected by vacuum filtration anddried in a vacuum oven at 70° C. overnight to provide Example 1 as awhite solid (1.62 g, 80%): mp 386-387° C.; TLC R_(f) (4:1CH₂Cl₂/-EtOAc)=0.22. ¹H NMR (300 MHz, CDCl₃) 5: 7.53 (d, J=7.7 Hz, 1H),7.38-7.08 (m,3H), 7.02 (s, 1H), 6.80 (s, 1H), 6.75-6.58 (m, 2H), 5.93(s, 2H), 4.32 (dd, J=11.9, 4.2 Hz, 1H), 4.13 (d, J=17.7 Hz, 1H), 3.98(d, J=17.7 Hz, 1H), 3.54 (dd, J=15.6, 4.3 Hz, 1H), 3.41 (s, 3H),3.12-2.87 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ: 165.5, 161.3, 148.2,148.0, 137.3, 131.6, 131.1, 125.9, 122.2, 119.6, 118.4, 109.1, 108.9,108.3, 107.6, 101.3, 52.0, 51.4, 50.9, 33.3, 29.9, 27.5 ppm; API MS m/z404 [C₂₃H₂₁N₃O₄+H]⁺; [α]_(D) ^(22° C.)=−^(346.0)° (c=1.0, DMSO). Anal.Calcd. for C₂₃H₂₁N₃O₄: C, 68.47; H, 5.25; N, 10.42. Found: C, 68.12, H,5.56; N, 10.05. The relative stereochemistry of Example 1 was confirmedto be the trans isomer by a series of NOE difference experiments(DMSO-d₆): no NOE enhancement from the C12a proton at 4.16 ppm to the C6proton at 6.98 ppm; no NOE enhancement from the C6 proton at 6.98 ppm tothe C6 proton at 4.16 ppm. The absolute stereochemistry of Example 1 wasconfirmed by an empirical circular dichroism experiment.

EXAMPLE 2

[0098](6R,12aR)-6-Benzo[1,3]dioxol-5-yl-2,7-dimethyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indole-1,4-dione

[0099] Example 2 was prepared from 1-methyl-D-tryptophan as depicted inthe following synthetic Scheme 2.

[0100] Preparation of 1-Methyl-D-tryptophan methyl ester hydrochloride(Intermediate 1)

[0101] Thionyl chloride (1.3 mL, 18.4 mmol) was added dropwise to asuspension of 1-methyl-D-tryptophan (2.0 g, 9.2 mmol) in MeOH (30 mL) at0° C. under a nitrogen blanket. The resulting mixture was warmed slowlyto room temperature and stirred for a total of 20 hours. The solvent wasremoved under reduced pressure and triturated with Et₂O (20 mL). Thesolids were collected by vacuum filtration, then dried in a vacuum ovenat 60° C. for 3 days to provide Intermediate 1 as an off-white powder,which was used without further purification (2.4 g, 96%): TLC R_(f) (1:2EtOAc/CHCl3)=0.22.

[0102] Preparation of cis-β-Carboline Intermediate 2

[0103] A suspension of Intermediate 1 (2.4 g, 8.9 mmol) and piperonal(1.5 g, 9.8 mmol) in IPA (25 mL) was stirred at reflux under a nitrogenblanket for 4 hours. The cooled mixture was diluted with IPA (20 mL),then the solid was removed by vacuum filtration. The filtrate wasconcentrated to afford a brown oil, which was purified by flash columnchromatography, eluting with EtOAc/CH₂Cl₂ (1:9), to provide theIntermediate 2 as a white solid, but not characterized (0.50 g, 16%):TLC R_(f) (1:2 EtOAc/—CH₂Cl₂)=0.84. The trans carboline also wasobtained as a white solid, but not characterized (1.6 g, 50%): TLC R_(f)(1:2)=0.76.

[0104] Preparation of cis-Chloroacetyl-β-carboline Intermediate 3

[0105] Chloroacetyl chloride (0.13 mL, 1.6 mmol) was added dropwise to asolution of Intermediate 2 (0.44 g, 1.2 mmol) and Et₃N (0.22 mL, 1.6mmol) in CH₂Cl₂ (20 mL) at 0° C. under a nitrogen blanket. The mixturewas slowly warmed to room temperature and stirred for 16 hours. Theresulting white suspension was diluted with CH₂Cl₂ (100 mL), washed withbrine (100 mL), dried over Na₂SO₄, and filtered. The solvent was removedunder reduced pressure to provide Intermediate 3 as a yellow foam, whichwas used without further purification (0.47 g): TLC R_(f) (1:2EtOAc/CHCl₂)=0.91.

[0106] Preparation of Example 2

[0107] A mixture of crude Intermediate 3 (0.46 g, 1.0 mmol) and CH₃NH₂(2.5 mL, 0.5 mmnol, 2.0 M in THF) in CH₃OH (20 mL) was heated at refluxunder a nitrogen blanket for 18 hours, after which the resulting orangesolution was cooled to room temperature. The solvent was removed underreduced pressure to provide a brown oil. This residue was purified byflash column chromatography, eluting with EtOAc/CHCl₃ (1:3), to provideExample 2 as an amber powder (0.3 g, 75% over two steps): mp 228-231°C.; TLC R_(f) (1:3 EtOAc/CHCl₃)=0.41. ¹H NMR (500 MHz, DMSO-d₆) δ: 7.68(d, J=7.9 Hz, 1H) , 7.08-7.13 (m, 3H) , 7.85 (d, J=8.1 Hz, 1H), 7.80 (s,1H), 6.75 (d, J=8.1 Hz, 1H), 6.30 (s, 1H), 5.89 (s, 1H), 5.87 (s, 1H),4.27-4.21 (m, 1H), 4.12 (d, J=17.4 Hz, 1H), 3.91 (d, J=17.4 Hz, 1H),3.80-3.75 (m, 1H), 3.30-3.24 (m, 1H), 3.03 (s, 3H); API MS m/z 404(C₂₃H₂₁N₃O₄+H)⁺; [α]_(D) ^(25° C.)=+10.0° (c=1.0, CHCl₃). Anal. Calcd.for C₂₃H₂₁N₃O₄: C, 66.98; H, 5.38; N, 10.19. Found: C, 67.26; H, 5.38;N, 9.83. The stereochemistry of Example 2 was confirmed to be thedesired cis isomer by a series of NOE difference experiments: a positiveNOE enhancement from the C12a proton at 4.24 ppm to the C6 proton at6.30 ppm; a positive NOE enhancement from the C6 proton at 6.30 ppm tothe C12a proton at 4.24 ppm.

EXAMPLE 3

[0108](6R,12aS)-6-Benzo[1,3]dioxol-5-yl-7-benzyl-2-methyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]prido[3,4-b]indole-1,4-dione

[0109] Example 3 was prepared in one step from compound (IV) byalkylation with benzyl bromide. Like Example 1, the basic reactionconditions resulted in complete epimerization of compound (IV).

[0110] A dried flask under a nitrogen blanket was charged with 2.05 g(5.26 mmol) of compound (IV) and dry THF, then cooled to 0° C. withstirring. NaH (0.32 g, 7.9 mmol) was added as a 60% oil dispersion inseveral portions. The reaction mixture was allowed to warm to ambienttemperature over 15 minutes, then 0.69 ml (5.8 mmol) benzyl bromide wasadded. After 20 hours, the reaction mixture was diluted with EtOAc,washed with 3% NaHSO₄, sat. NaHCO₃, and brine, dried over Na₂SO₄,filtered, and the solvent stripped on a rotavapor. The resulting oil waspurified by flash chromatography (4.8×23 cm, CH₂Cl₂/EtOAc/MeOH (90:10:1)to yield after drying in vacuo 2.14 g (85% yield) of a white amorphoussolid: mp 110-145° C. ¹H NMR (DMSO-d₆) δ: 7.59 (d, J=7.6, 1H), 7.40 (d,J=7.9, 1H), 7.26-7.07 (m, 5H), 6.88-6.80 (m, 4H), 6.71 (s, 1H), 6.58 (d,J=8.0, 1H), 6.00 (d, J=10.8, 2H), 5.36 (d, J=16.8, 1H), 4.75 (d, J=16.8,1H), 4.23 (d, J=17.6, 1H), 4.12 (d of d, J₁=11.7, J₂=4.0, 1H), 3.99 (d,J=17.6, 1H), 3.35 (d of d, J₁=14.1, J₂ obscured by water peak, 1H), 3.01(d of d, J₁=12.1, J₂=15.1, 1H), 2.83 (s, 3H); TLC R_(f)(CH₂Cl₂/EtOAc/MeOH) (90:10:1)=0.31; MS m/z 502 (M+Na); [α]_(D)^(25° C.)=−230.2 (c=0.1, DMSO); Anal. Calcd. for C₂₉H₂₅N₃O₄: C, 72.64;H, 5.25; N, 8.76. Found: C, 72.46; H, 5.40; N, 8.42. Transstereochemistry was confirmed by HMQC and NOE experiments: HMQC assigns6.80 ppm singlet to C6 proton; positive NOE enhancement observed fromC12a (4.12 ppm) to pendent aryl protons at 6.58 ppm and 6.71 ppm and notto C6, and no NOE observed from C6 to C12a.

EXAMPLE 4

[0111](6R,12aR)-7-Acetyl-6-benzo[1,3]dioxol-5-yl-2-methyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione

[0112] Example 4 was prepared by aceylating compound (IV) using aceticanhydride.

[0113] Compound (IV) (2.01 g, 5.16 mmol), 4-dimethylaminopyridine (0.946g, 7.74 mmol), and Ac₂O (0.97 ml, 10 mmol) were slurried in THF in a dryflask under a nitrogen blanket and stirred magnetically. The reactionwas monitored by TLC. After 24 hours, an additional 0.97 ml Ac₂O wasadded. After 7 days, the reaction was quenched by dilution with EtOAc,and aqueous work-up (washed with sat. NaHCO₃, 1 N HCl, and sat. NaCl,dried over Na₂SO₄, filtered, and stripped on a rotavapor). The residuewas purified by flash chromatography (4.8×22 cm, CH₂Cl₂/-EtOAc/MeOH)(90:10:1) to yield after drying in vacuo 1.87 g (84%) of a whiteamorphous solid: mp 145-159° C. ¹H NMR (DMSO-d₆) δ: 7.85 (d, J=6.8, 1H),7.78 (d, J=6.8, 1H), 7.37-7.33 (m, 2H), 7.09 (s, 1H), 6.78 (s, 1H),6.71- (d, J=8.1, 1H), 6.64 (d, J=8.1, 1H), 5.92 (d, J=4.4, 2H), 4.42 (dof d, J₁=11.7, J₂=4.5, 1H), 4.21 (d, J=17.0, 1H), 3.93 (d, J=17.1, 1H),3.55 (d of d, J₁=16.4, J₂=4.7, 1H), 2.96 (d of d, J₁=16.4, J₂=12.6, 1H),2.90 (s, 3H), 2.71 (s, 3H) ; TLC R_(f) (CH₂Cl₂/EtOAc/MeOH)(90:10:1)=0.22; MS m/z 454 (M+Na); Anal. Calcd. for C₂₄H₂₁N₃O₅, 0.33H₂O: C, 65.91; H, 4.99; N, 9.61. Found: C, 65.95; H, 4.75; N, 9.64. Cisstereochemistry was confirmed by NOE experiments: Positive NOEenhancements observed from C12a (4.42 ppm) to C6 (7.09 ppm) and from C6to C12a.

EXAMPLE 5

[0114]

[0115] Compounds of the present invention can be formulated into tabletsfor oral administration. For example, a compound of formula (I) can beformed into a dispersion with a polymeric carrier by the coprecipitationmethod set forth in WO 96/38131, incorporated herein by reference. Thecoprecipitated dispersion then can be blended with excipients, thenpressed into tablets, which optionally are film-coated.

[0116] The compounds of structural formula (I) were tested for anability to inhibit PDE5. The ability of a compound to inhibit PDE5activity is related to the IC50 value for the compound, i.e., theconcentration of inhibitor required for 50% inhibition of enzymeactivity. The IC50 value for compounds of structural formula (I) weredetermined using recombinant human PDE5.

[0117] The compounds of the present invention typically exhibit an IC₅₀value against recombinant human PDE5 of less than about 50 μM, andpreferably less than about 25 μM, and more preferably less than about 15μm. The compounds of the present invention typically exhibit an IC₅₀value against recombinant human PDE5 of less than about 1 μM, and oftenless than about 0.25 μM. To achieve the full advantage of the presentinvention, a present PDE5 inhibitor has an IC50 of about 0.1 nM to about15 μM.

[0118] The production of recombinant human PDEs and the IC₅₀determinations can be accomplished by well-known methods in the art.Exemplary methods are described as follows:

EXPRESSION OF HUMAN PDEs

[0119] Expression in Saccharomyces cerevisiae (Yeast)

[0120] Recombinant production of human PDE1B, PDE2, PDE4A, PDE4B, PDE4C,PDE4D, PDE5, and PDE7 was carried out similarly to that described inExample 7 of U.S. Pat. No. 5,702,936, incorporated herein by reference,except that the yeast transformation vector employed, which is derivedfrom the basic ADH2 plasmid described in Price et al., Methods inEnzymology, 185, pp. 308-318 (1990), incorporated yeast ADH2 promoterand terminator sequences and the Saccharomyces cerevisiae host was theprotease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with theAmerican Type Culture Collection, Manassas, Va., under accession numberATCC 74465. Transformed host cells were grown in 2X SC-leu medium, pH6.2, with trace metals, and vitamins. After 24 hours, YEPmedium-containing glycerol was added to a final concentration of 2XYET/3% glycerol. Approximately 24 hr later, cellswere harvested, washed,and stored at −70° C.

HUMAN PHOSPHODIESTERASE PREPARATIONS

[0121] Phosphodiesterase Activity Determinations

[0122] Phosphodiesterase activity of the preparations was determined asfollows. PDE assays utilizing a charcoal separation technique wereperformed essentially as described in Loughney et al. (1996). In thisassay, PDE activity converts [32P]cAMP or [32P]cGMP to the corresponding[32P]5′-AMP or [32P]5′-GMP in proportion to the amount of PDE activitypresent. The [32P]5′-AMP or [32P]5′-GMP then was quantitativelyconverted to free [32P]phosphate and unlabeled adenosine or guanosine bythe action of snake venom 5′-nucleotidase. Hence, the amount of[32P]phosphate liberated is proportional to enzyme activity. The assaywas performed at 30° C. in a 100 μL reaction mixture containing (finalconcentrations) 40 mM Tris HCl (pH 8.0), 1 μM ZnSO₄, 5 mM MgCl₂, and 0.1mg/mL bovine serum albumin (BSA). PDE enzyme was present in quantitiesthat yield <30% total hydrolysis of substrate (linear assay conditions).The assay was initiated by addition of substrate (1 mM [32P]cAMP orcGMP), and the mixture was incubated for 12 minutes. seventy-five (75)μg of Crotalus atrox venom then was added, and the incubation wascontinued for 3 minutes (15 minutes total). The reaction was stopped byaddition of 200 μL of activated charcoal (25 mg/mL suspension in 0.1 MNaH₂PO₄, pH 4). After centrifugation (750 X g for 3 minutes) to sedimentthe charcoal, a sample of the supernatant was taken for radioactivitydetermination in a scintillation counter and the PDE activity wascalculated.

[0123] Purification of PDE5. from S. cerevisiae

[0124] Cell pellets (29 g) were thawed on ice with an equal volume ofLysis Buffer (25 mM Tris HCl, pH 8, 5 mM MgCl₂, 0.25 mM DTT, 1 mMbenzamidine, and 10 μM ZnSO₄). Cells were lysed in a Microfluidizer®(Microfluidics Corp.) using nitrogen at 20,000 psi. The lysate wascentrifuged and filtered through 0.45 μm disposable filters. Thefiltrate was applied to a 150 mL column of Q SEPHAROSE® Fast-Flow(Pharmacia). The column was washed with 1.5 volumes of Buffer A (20 mMBis-Tris Propane, pH 6.8, 1 mM MgCl₂₁ 0.25 mM DTT, 10 μM ZnSO₄) andelutedwith a step gradient of 125 mM NaCl in Buffer A followed by alinear gradient of 125-1000 mM NaCl in Buffer A. Active fractions fromthe linear gradient were applied to a 180 mL hydroxyapatite column inBuffer B (20 mM Bis-Tris Propane (pH 6.8), 1 mM MgCl₂, 0.25 mM DTT, 10μM ZnSO₄, and 250 mM KCl). After loading, the column was washed with 2volumes of Buffer B and eluted with a linear gradient of 0-125 mMpotassium phosphate in Buffer B. Active fractions were pooled,precipitated with 60% ammonium sulfate, and resuspended in Buffer C (20mM Bis-Tris Propane, pH 6.8, 125 mM NaCl, 0.5 mM DTT, and 10 μM ZnSO₄).The pool was applied to a 140 mL column of SEPHACRYL® S-300 HR andeluted with Buffer C. Active fractions were diluted to 50% glycerol andstored at −20° C.

[0125] The resultant preparations were about 85% pure by SDS-PAGE. Thesepreparations had specific activities of about 3 μmol cGMP hydrolyzed perminute per milligram protein.

[0126] Inhibitory Effect on CGMP-PDE

[0127] cGMP-PDE activity of compounds of the present invention wasmeasured using a one-step assay adapted from Wells et al., Biochim.Biophys. Acta, 384, 430 (1975). The reaction medium contained 50 mMTris-HCl, pH 7.5, 5 mM magnesium acetate, 250 μg/ml 5′-Nucleotidase, 1mM EGTA, and 0.15 μM 8-[H³]-cGMP. Unless otherwise indicated, the enzymeused was a human recombinant PDE5 (ICOS Corp., Bothell, Wash.).

[0128] Compounds of the invention were dissolved in DMSO finally presentat 2% in the assay. The incubation time was 30 minutes during which thetotal substrate conversion did not exceed 30%.

[0129] The IC₅₀ values for the compounds examined were determined fromconcentration-response curves typically using concentrations rangingfrom 10 nM to 10 μM. Tests against other PDE enzymes using standardmethodology showed that compounds of the invention are selective for thecGMP-specific PDE enzyme.

[0130] Biological Data

[0131] The compounds according to the present invention were typicallyfound to exhibit an IC₅₀ value of less than 500 nM (i.e., 0.5 μM). Invitro test data for representative compounds of the invention is givenin the following table: TABLE 1 In vitro Results Example PDE5 IC₅₀ (μM)1 0.124 2 0.571 3 0.313 4 0.007

[0132] Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof, and, therefore, only such limitations should be imposedas are indicated by the appended claims.

What is claimed is:
 1. A compound having a formula

wherein R⁰, independently, is selected from the group consisting of haloand C₁₋₆alkyl; R¹ is selected from the group consisting of hydro,C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, haloC₁₋₆alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, arylC₁₋₃alkyl, C₁₋₃alkylenearyl, andheteroarylC₁₋₃alkyl; R² is selected from the group consisting of anoptionally substituted monocyclic aromatic ring selected from the groupconsisting of benzene, thiophene, furan, and pyridine, and an optionallysubstituted bicyclic ring

wherein the fused ring A is a 5- or 6-membered ring, saturated orpartially or fully unsaturated, and comprises carbon atoms andoptionally one or two heteroatoms selected from oxygen, sulfur, andnitrogen; R³ is selected from the group consisting of hydro andC₁₋₆alkyl, or R¹ and R³ together form a 3- or 4-membered alkyl oralkenyl chain component of a 5- or 6-membered ring; R⁴ is selected fromthe group consisting of C₁₋₆alkyl, C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl,C₂₋₆-alkenyl, C₁₋₃alkylenearyl, arylC₁₋₃alkyl, heteroarylC₁₋₃alkyl,C(═O)R^(a), aryl, heteroaryl, C(═O)OR^(a), C(═O)—NR^(a)R^(b),C(═S)NR^(a)R^(b), SO₂R^(a), SO₂OR^(a), SO₂NR^(a)R^(b), S(═O)R^(a),S(═O)NR^(a)R^(b), C(═O)NR^(a)C₁₋₄alkyleneOR^(a),C(═O)NR^(a)C₁₋₄alkyleneHet, C(═O)C₁₋₄alkylenearyl,C(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkylenearyl substituted with one ormore Of SO₂NR^(a)R^(b), NR^(a)R^(b), C(═O)OR^(a), NR^(a)SO₂CF₃, CN, NO₂,C(═O)R^(a), OR^(a), C₁₋₄alkyleneNR^(a)R^(b), andOC₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneHet, C₁₋₄alkyleneC(═O)C₁₋₄alkylenearyl, C₁₋₄alkyleneC(═O)C₁₋₄alkyleneheteroaryl,C₁₋₄alkyleneC(═O)Het, C₁₋₄alkyleneC(═O)NR^(a)R^(b), C₁₋₄alkyleneOR^(a),C₁₋₄alkyleneNR^(a)C (═O)R^(a), C₁₋₄alkyleneOC₁₋₄alkyleneOR^(a),C₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneC(═O)OR^(a), andC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a); Het represents a 5- or 6-memberedheterocyclic ring, saturated or partially or fully unsaturated,containing at least one heteroatom selected from the group consisting ofoxygen, nitrogen, and sulfur, and optionally substituted with C₁₋₄alkylor C(═O)OR^(a); R^(a) is selected from the group consisting of hydro,C₁₋₆alkyl, aryl, arylC₁₋₃alkyl, C₁₋₃alkylenearyl, heteroaryl,heteroarylC₁₋₃alkyl, and C₁₋₃alkyleneheteroaryl; R^(b) is selected fromthe group consisting of hydro, C₁₋₆alkyl, aryl, heteroaryl,arylC₁₋₃alkyl, heteroarylC₁₋₃alkyl, C₁₋₃alkyleneN(R^(a))₂,C₁₋₃alkylenearyl, C₁₋₃alkyleneHet, haloC₁₋₃alkyl, C₃₋₈cycloalkyl,C₃₋₈heterocycloalkyl, C₁₋₃alkyleneheteroaryl, C₁₋₃alkyleneC(═O)OR^(a),and C₁₋₃alkyleneC₃₋₈heterocycloalkyl; or R^(a) and R^(b) are takentogether to form a 5- or 6-membered ring, optionally containing at leastone heteroatom; q is 0, 1, 2, 3, or 4; and pharmaceutically acceptablesalts and hydrates thereof.
 2. The compound of claim 1 represented bythe formula

and pharmaceutically acceptable salts and hydrates thereof.
 3. Thecompound of claim 1 wherein q is 0 or R⁰ is selected from the groupconsisting of halo and C₁₃alkyl.
 4. The compound of claim 1 wherein R²is the optionally substituted bicyclic ring


5. The compound of claim 4 wherein R² is

and wherein n is an integer 1 or 2, and G, independently, are C(R^(a))₂,O S, or NR^(a).
 6. The compound of claim 1 wherein R² is selected fromthe group consisting of


7. The compound of claim 1 wherein the R⁴ group is selected from thegroup consisting of C₁₋₆-alkyl, C(═O)R^(a), C(═O)OR^(a), SO₂NR^(a)R^(b),aryl, heteroaryl, C₁₋₄alkyleneHet, C₁₋₄alkyleneheteroaryl,C₁₋₄alkylenearyl, C₁₋₄alkyleneC(═O)C₁₋₄alkylenearyl,C₁₋₄alkyleneC(═O)OR^(a), C₁₋₄alkyleneC(═O)NR^(a)R^(b),C₁₋₄alkyleneC(═O)Het, C₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneOR^(a), andC₁₋₄alkyleneNR^(a)C(═O)R^(a).
 8. The compound of claim 7 wherein R⁴ isselected from the group consisting of C₁₋₆alkyl, C(═O)R^(a),SO₂NR^(a)R^(b); C₁₋₄alkyleneHet, wherein Het is selected from the groupconsisting of piperazinyl, morpholinyl, pyrrolidinyl, pyrrolidonyl,tetrahydrofuran, piperidinyl,

C₁₋₄alkyleneC₆H₅, optionally substituted with one to three groupsselected from the group consisting of C(═O)OR^(a), NR^(a)R^(b),NR^(a)SO₂CF₃, SO₂N^(a)R^(b), CN, OR^(a), C(═O)R^(a),C₁₋₄alkyleneNR^(a)R^(b), nitro, OC₁₋₄alkylenearyl, andOC₁₋₄alkyleneNR^(a)R^(b); C₁₋₄alkyleneC(═O)benzyl; C₁₋₄alkyleneOR^(a);C₁₋₄alkyleneC(═O)OR^(a); C₁₋₄alkyleneC(═O)NR^(a)R^(b); C₆H₅;C1-4alkyleneNR^(a)R^(b); and C₁₋₄alkyleneNHC(═O)R^(a).
 9. The compoundof claim 8 wherein R⁴ is selected from the group consisting ofC₁₋₆alkyl, C₁₋₄alkylenearyl, C(═O)R^(a), and SO₂NR^(a)R^(b).
 10. Thecompound of claim 1 wherein q is 0 or R⁰ is halo or methyl; R¹ isselected from the group consisting of hydrogen, C₁₋₆alkyl, andhaloC₁₋₆alkyl; R² is selected from the group consisting of

R³ is C₁₋₆alkyl; and R⁴ is selected from the group consisting ofC₁₋₆alkyl, C(═O)R^(a), SO₂NR^(a)R^(b), C₁₋₄alkylenearyl,C₁₋₄alkyleneC(═O)OR^(a), C(═O)OR^(a), C₁₋₄alkyleneNR^(a)R^(b), andC₁₋₄alkyleneOR^(a).
 11. The compound of claim 10 wherein q is 0, R¹ ismethyl, R³ is hydro, and R⁴ is selected from the group consisting ofmethyl, benzyl, C(═O)CH₃, SO₂N(CH₃)₂, CHO, C₂H₅, CH(CH₃)₂,(CH₂)₄C(═O)OH, C(═O)OCH₃, CH₂NHCH₂C₆H₅, CH₂NH₂, and CH₂OH.
 12. Thecompound selected from the group consisting of(6R,12aS)-6-benzo[1,3]dioxol-5-yl-2,7-dimethyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione;(6R,12aR)-6-benzo[1,3]dioxol-5-yl-2,7-dimethyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione;(6R,12aS)-6-benzo[1,3]dioxol-5-yl-7-benzyl-2-methyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione;and(6R,12aR)-7-acetyl-6-benzo[1,3]dioxol-5-yl-2-methyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione;and pharmaceutically acceptable salts and solvates thereof.
 13. Acompound having the formula:


14. A pharmaceutical composition comprising a compound of claim 1,together with a pharmaceutically acceptable diluent or carrier.
 15. Amethod of treating a male or female animal in the treatment of acondition where inhibition of a cGMP-specific PDE is of a therapeuticbenefit comprising treating said animal with an effective amount of apharmaceutical composition comprising a compound of claim 1, togetherwith a pharmaceutically acceptable diluent or carrier.
 16. The method ofclaim 15 wherein the condition is male erectile dysfunction.
 17. Themethod of claim 16 wherein the treatment is an oral treatment.
 18. Themethod of claim 15 wherein the condition is female sexual arousaldisorder.
 19. The method of claim 18 wherein the treatment is an oraltreatment.
 20. The method of claim 15 wherein the condition is selectedfrom the group consisting of stable angina, unstable angina, variantangina, hypertension, pulmonary hypertension, chronic obstructivepulmonary disease, malignant hypertension, pheochromocytoma, acuterespiratory distress syndrome, congestive heart failure, acute renalfailure, chronic renal failure, atherosclerosis, a condition of reducedblood vessel patency, a peripheral vascular disease, a vasculardisorder, thrombocythemia, an inflammatory disease, myocardialinfarction, stroke, bronchitis, chronic asthma, allergic asthma,allergic rhinitis, glaucoma, peptic ulcer, a gut motility disorder,postpercutaneous transluminal coronary angioplasty, carotid angioplasty,post-bypass surgery graft stenosis, osteoporosis, preterm labor, benignprostatic hypertrophy, and irritable bowel syndrome.
 21. A method oftreating a condition where inhibition of a cGMP-specific PDE is oftherapeutic benefit, in a human or a nonhuman animal body, comprisingadministering to said body a therapeutically effective amount of acompound of claim
 1. 22. A method for the curative or prophylactictreatment of male erectile dysfunction or female sexual arousaldisorder, comprising administration of an effective dose of a compoundof claim 1, and pharmaceutically acceptable salts and solvates thereof,to an animal.
 23. Use of a compound of claim 1 for the manufacture of amedicament for the curative or prophylactic treatment-of a conditionwhere inhibition of a cGMP-specific PDE is of a therapeutic benefit.